Ce travail a été financé par des subventions à LKM-K de la National Institutes of Health / National Cancer Institute (R01CA129822 et R01CA140995) national. Dr Medina-Kauwe remercie C. Rey, M. M-Kauwe et D. Revetto d&#39;un soutien continu.

1. Livraison systémique in vivo <ol><li> Mélanger assez Herdox avec une solution saline stérile à assimiler 0,2 ml d&#39;une dose de 0,004 mg / kg de Herdox par injection pour un 6-8 semaines d&#39;âge nu / nu souris portant des tumeurs sous-cutanées bilatérales de xénogreffes de flanc. </li><li> Tirer doucement le mélange Herdox dans une seringue à insuline 3/10 cc équipé d&#39;une aiguille 29G, en évitant les bulles. </li><li> L&#39;anesthésie est induite par une brève exposition isoflurane dans une chambre d&#39;induction équipé d&#39;un système d&#39;évacuation des gaz (débit d&#39;oxygène: 0,5-1 L / min, la concentration d&#39;isoflurane: 3-4% (ou moins). </li><li> Injecter la totalité du mélange dans la veine de la queue d&#39;une souris anesthésiée (0,2 ml par injection). Injection IV peut également être effectuée dans un sobre, souris non anesthésiées. </li><li> Répéter les injections sur la même souris pendant six jours plus séquentielles, une fois par jour. </li></ol> 2. Fluorescence Imaging in vivo L&#39;accumulation de Herdox fluorescence dans les tumeurs peuventêtre détectable par le dernier jour de l&#39;injection (Jour 7) en utilisant un imageur multimode. Les procédures ci-dessous reposent sur ​​l&#39;utilisation d&#39;un système de macro-illumination et de détection personnalisée (Figure 1) 8. <ol><li> Allumez le Multimode En imageur optique in vivo. </li><li> Sélectionnez un filtre passe-bande d&#39;émission (590 nm ± 30 nm) adapté à la détection par fluorescence doxorubicine. </li><li> Activer le laser argon-krypton et de placer un filtre passe-bande d&#39;excitation (488 nm ± 10 nm) dans le chemin optique du laser. </li><li> Allumez le système d&#39;anesthésie et ensuite placer une souris dans la chambre anesthésiant (débits d&#39;oxygène: 0,5-1 L / min, la concentration d&#39;isoflurane: 3-4% (ou moins) équipés d&#39;un système d&#39;évacuation des gaz. </li><li> Transférer la souris de la chambre anesthésiant à la chambre de l&#39;imagerie de la Multimode In vivo imageur optique lorsque la souris est anesthésiée. </li><li> Placer un cône de nez sur le nez de la souris et ouvrir le flux d&#39;administrer anesthésie continuesia cours acquisition d&#39;images (débits d&#39;oxygène: 0,5-1 L / min, la concentration d&#39;isoflurane: 2-3% (ou moins). </li><li> Acquérir les images de fluorescence en utilisant un temps d&#39;exposition de 5-15 sec. </li><li> Procéder à l&#39;analyse d&#39;image et de traitement, y compris la correction de fond ou de réglage du contraste. </li></ol> 3. Fluorescence Imaging Ex vivo Herdox fluorescence peut être visualisé dans les tumeurs et des organes spécifiques (y compris le foie, les reins, la rate, le cœur et les muscles squelettiques) récoltées à partir des souris euthanasiés à 24 heures après la dernière injection (Jour 7) de Herdox. <ol><li> Allumez le Multimode En imageur optique in vivo. </li><li> Sélectionnez un filtre passe-bande d&#39;émission (580 nm ± 20 nm) pour la détection par fluorescence doxorubicine. </li><li> Activer le laser argon-krypton et de placer un filtre passe-bande d&#39;excitation (488 nm ± 10 nm) dans le chemin optique du laser. </li><li> Placer les tumeurs et les organes spécifiques disposés sur une boîte de Petri dans la chambre d&#39;imagerie of le Multimode En imageur optique in vivo. </li><li> Acquérir les images de fluorescence de tissus en utilisant un temps d&#39;exposition de 5-15 sec. Un exemple de première acquisition d&#39;image par fluorescence est représenté en figure 2a. Répétez la même chose en utilisant une boîte de Petri vide, qui servira de toile de fond (Figure 2b). </li><li> Procéder à l&#39;analyse d&#39;image et de traitement, y compris la correction de fond ou de réglage du contraste. Un exemple d&#39;une image corrigée est illustré à la figure 2c, résultant de la soustraction de la figure 2b de la figure 2a. </li></ol> 4. Dans l&#39;imagerie confocale in situ des tumeurs Dans l&#39;imagerie confocale in situ permet la détection et l&#39;analyse de l&#39;accumulation de tumeur Herdox au niveau cellulaire. <ol><li> Tourner sur un Leica SPE microscope confocal. </li><li> Sélectionner une lumière laser 488 nm pour l&#39;excitation de la doxorubicine et l&#39;émission des longueurs d&#39;onde (560 à 620 nm) pour la doxorubicinela détection de fluorescence. </li><li> Sélectionnez un objectif 40X ou 63X et l&#39;huile d&#39;immersion goutte sur l&#39;objectif. </li><li> Extrait tumeurs fraîches de souris euthanasiés qui ont déjà reçu des traitements Herdox et se moquent comme décrit dans la procédure 2. </li><li> Placer les tumeurs sur une boîte de Pétri sur la glace pour éviter la dégradation du tissu, puis transférer les tumeurs à une chambre Delta T pour l&#39;imagerie confocale. </li><li> Acquérir les images confocale des tumeurs à des profondeurs focales séquentiels (taille d&#39;étape: 1 pm, épaisseur: 20 pm). Un exemple d&#39;images successivement acquises le long de l&#39;axe z est illustré à la figure 3, panneau de gauche. </li><li> Effectuer intensité maximale z-projection des images. Une projection d&#39;intensité maximum de z-stack images est illustré à la figure 3, panneau de droite. </li><li> Calcul des intensités de fluorescence moyenne de l&#39;intensité maximale. Z - images de projection moyenne des intensités de fluorescence des images sur le champ de vue global était calculerd utiliser ImageJ. </li></ol> 5. Imagerie spectrale et radiométrique Analyse Imagerie spectrale et radiométrique analyse permet la discrimination entre Dox fluorescence et autofluorescence. <ol><li> Puissance sur un microscope confocal à balayage laser fluorescence. </li><li> L&#39;acquisition de 15 images des tumeurs traitées par Herdox et non traitée, à une profondeur spécifiée dans la plage spectrale de 510 à 650 nm, avec une taille de pas de 10 nm, et la lumière d&#39;excitation à 488 nm en utilisant un microscope confocal Leica SPE. </li><li> Préparer une solution à 100 uM de la doxorubicine. </li><li> Effectuer imagerie spectrale de la solution de doxorubicine de 100 pm pour obtenir la signature spectrale pure de Dox fluorescence (gamme spectrale: 510 à 650 nm, une taille de pas: 10 nm). Les résultats typiques de l&#39;acquisition d&#39;images à partir de l&#39;imagerie spectrale et résultant spectre de fluorescence représentées sous forme de graphique sont présentés dans la figure 4. </li><li> Acquérir la signature spectrale autofluorescence d&#39;une image cube (gamme spectrale: 510 à 650 nm, une taille de pas: 10 nm) obtenue par l&#39;imagerie spectrale des tumeurs non traitées. Les résultats typiques de l&#39;acquisition d&#39;images à partir de l&#39;imagerie spectrale et résultant spectre de fluorescence représentées sous forme de graphique sont présentés dans la figure 5. </li><li> Générer quatre signatures spectrales de référence (autofluorescence pure, 0.1.doxorubin +0,9. Autofluorescence, 0,2. Doxorubicine +0,8. Autofluorescence, 0.3.doxorubin +0,7. Autofluorescence) en utilisant le programme que nous avons mis au point 9. Une courbe typique montrant quatre signatures spectrales de référence est montré dans la figure 6. </li><li> Effectuer classification spectrale des images tel que défini par la référence signatures spectrales par mesure de distance euclidienne en utilisant le programme que nous avons précédemment développé 9. </li><li> Effectuer déconvolution spectrale linéaire de ces images en utilisant un programme spectrale de démixage (plug-in dans ImageJ), nous avons développé 7, 10, par rapport à l&#39;imagerie spectrale quotientométrique et d&#39;analyse. Exemple de séparer fluorescence Herdox de autofluorescence par déconvolution spectrale linéaire est illustré à la figure 7. </li></ol>

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L&#39;auteur, Daniel Farkas, est président du Molecular Imaging Spectral. Les auteurs n&#39;ont aucun conflit d&#39;intérêts.

Dox fluorescence peut être détectable in vivo en utilisant l&#39;imageur multimode lorsque les tumeurs sont sous-cutanée. Cependant, la dose thérapeutiquement efficace d&#39;Herdox (0,004 mg / kg) est inférieur au seuil de détection après une seule dose. En revanche, après 7 injections quotidiennes (1x/jour pendant 7 jours), l&#39;accumulation tumorale et la rétention de la particule est suffisante pour permettre la visualisation de Dox fluorescence. Il est essentiel quand ...

Ciblage tumoral de la chimiothérapie a le potentiel d&#39;éliminer les cellules cancéreuses à dose plus faible par rapport aux médicaments non ciblées parce que plus de la thérapie livré peut s&#39;accumuler à sa destination plutôt que de distribuer des tissus non tumoraux. Comme cette dernière situation diluerait sur l&#39;efficacité du médicament et donc nécessiter des doses plus élevées pour être efficace, de ciblage tumoral présente des avantages thérapeutiques et de sécurité par rapport au traitement non ciblée standard. Cibler la chimiothérapie par encapsulation dans des nanoparticules auto-assemblés permet au médicament de rester sans modification chimique contrairement aux médicaments qui sont liés de manière covalente à des molécules de ciblage. Comme tel lien a le potentiel de modifier l&#39;activité à la fois la drogue et la molécule de ciblage, l&#39;assemblage non covalente permet la puissance du médicament doit être conservé. Nous avons précédemment montré que le roman à trois composants, complexe auto-assemblée, Herdox, cible HER2 + tumeurs <em&gt; In vivo et suscite l&#39;ablation de tumeurs croissance tout en épargnant les tissus normaux, y compris le cœur 1. Herdox est formé par des interactions non covalentes entre la protéine de liaison au récepteur cellulaire pénétration, HerPBK10, et l&#39;agent de chimiothérapie, la doxorubicine (Dox), par l&#39;intermédiaire d&#39;un petit segment de liaison d&#39;acide nucléique. HerPBK10 se lie au récepteur du facteur de croissance épidermique humain (HER) et déclenche l&#39;endocytose médiée par un récepteur 4.2, alors que la pénétration de la membrane de l&#39;endosome est accompli grâce à l&#39;incorporation de l&#39;adénovirus-base du penton dérivé protéine de capside 4-6. Un domaine chargé positivement sur ​​de la protéine permet d&#39;acide nucléique de liaison 4, 5, par lequel l&#39;ADN-Dox intercalaire peut être transportée pour l&#39;administration ciblée. Électrophile, l&#39;intercalation, et, éventuellement, des interactions d&#39;oligomérisation des protéines de faciliter l&#39;auto-assemblage en particules rondes de 10 à 20 nm qui sont stables dans le sang et en cas de stockage prolongé à des températures différentes 1. Préférentiel visant à HER2 + cellules tumorales est facilitée par le renforcement affinité du ligand lorsque HER2 est élevé. Nos études antérieures ont montré que l&#39;administration systémique de rendements Herdox accumulation préférentielle dans les tumeurs de plus de tissu non tumoral et par rapport aux non ciblés Dox 1, et la pénétration dans les cellules tumorales in vivo 7. Nous avons observé que les rejets Herdox Dox après l&#39;entrée des cellules tumorales, permettant l&#39;accumulation Dox dans le noyau 1. Tumeur accumulation semble être en corrélation avec le niveau du récepteur, comme relativement faibles tumeurs HER2-exprimant accumulent moins Herdox par rapport à ceux ayant des niveaux relativement élevés de HER2 1. En outre, la concentration de la mort cellulaire efficace présente une corrélation inverse avec HER2 affichage sur des lignées cellulaires tumorales exprimant la surface des cellules différentes HER2 niveaux 1. Herdox présente un avantage thérapeutique et la sécurité sur Dox non ciblé, que l&#39;homicide tumeur survient au cours dose 10 fois inférieure par rapport à la untarstances visées médicament et ne donne aucun effet négatif détectable sur le cœur (détectée par échocardiographie et histologiques tache) ou le foie (détectée par TUNEL tache) tissus, contrairement aux non ciblée Dox 1. En dépit de sa dérivation à partir d&#39;une protéine de capside virale, HerPBK10 présente aucune immunogénicité décelable à des niveaux thérapeutiques 2. Tandis que les anticorps pré-existants à l&#39;ensemble de l&#39;adénovirus peuvent reconnaître HerPBK10, ils sont incapables d&#39;empêcher la liaison cellulaire 2. Volume tumoral mesuré au fil du temps est une méthode standard d&#39;évaluation de l&#39;efficacité thérapeutique des thérapies ciblées, et a été utilisée pour évaluer l&#39;efficacité thérapeutique de Herdox. Complétant cette approche avec imagerie in vivo et ex vivo de l&#39;intensité de fluorescence nous a permis de mieux évaluer l&#39;efficacité du ciblage 7. Nous avons spécifiquement intégré dans l&#39;imagerie confocale in situ des tumeurs excisées avec l&#39;analyse spectrale de Dox fluorescence pour vérifier que Herdox past seulement accumulé à des tumeurs in vivo, mais pénètre dans les cellules tumorales et Dox livré dans le cytoplasme et le noyau 7. L&#39;analyse spectrale de plus nous a permis de distinguer Dox fluorescence de autofluorescence 7. Ici, nous démontrons plus en détail notre approche pour évaluer Herdox in vivo après administration systémique, et surtout, pour évaluer le ciblage par des méthodes et des analyses d&#39;imagerie multimodes.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

La nanoparticule HER2 + tumeur-spécifique, Herdox, présente une accumulation de tumeur préférentiel et l&#39;ablation de tumeurs se développer dans un modèle animal de cancer de HER2 +. Herdox est formée par des non-covalente d&#39;auto-assemblage d&#39;une protéine de pénétration cellulaire ciblé tumeur avec l&#39;agent de chimiothérapie, la doxorubicine, par l&#39;intermédiaire d&#39;un petit segment de liaison d&#39;acide nucléique. Une combinaison de électrophile, intercalation, et les interactions d&#39;oligomérisation faciliter l&#39;auto-assemblage en particules rondes 10-20 nm. Herdox présente une stabilité dans le sang, ainsi que dans un stockage prolongé à des températures différentes. Administration systémique de Herdox chez les souris porteuses de tumeurs résultats dans la mort des cellules tumorales, sans effets néfastes décelables à un tissu non tumoral, y compris le cœur et le foie (qui subissent des dommages marquée par la doxorubicine non ciblée). HER2 facilite élévation de ciblage pour des cellules exprimant le récepteur du facteur de croissance épidermique humain, d&#39;où les tumeurs présentant des taux élevés de HER2 présentent une plus grande accumulation de Herdox rapport aux cellules expressionchanter des niveaux inférieurs, à la fois in vitro et in vivo. Imagerie de fluorescence associée à l&#39;analyse spectrale confocale et in situ a permis de vérifier dans la tumeur in vivo ciblage et la pénétration des cellules tumorales de Herdox après l&#39;accouchement systémique. Ici, nous détaillons nos méthodes pour évaluer le ciblage tumoral par l&#39;imagerie multimodale après l&#39;accouchement systémique.

La figure 1 montre le prototype d&#39;imageur optique in vivo, qui a été construit dans le but d&#39;acquisition d&#39;image en de multiples modalités, y compris l&#39;intensité de fluorescence, spectrale, durée de vie, 2-photon, confocale intra-vital, et l&#39;imagerie par bioluminescence in. En outre, la caméra haute sensibilité refroidie et lignes laser haute puissance intégrée dans ce système rendements contraste des images de fluorescence élevés par rapport aux systèm...

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Cet article détaille les procédures d&#39;analyse d&#39;imagerie optique de la nanoparticule tumeur ciblée, Herdox. En particulier, l&#39;utilisation de détail du dispositif d&#39;imagerie multimode pour détecter le ciblage tumoral et l&#39;évaluation de la pénétration tumeur est décrit ici.

Analyse de la protéine virale ciblée nanoparticules administrées à HER2 + Tumeurs

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

analysis-targeted-viral-protein-nanoparticles-delivered-to-her2

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10.9K vues.  University of Southern California. Cet article détaille les procédures d'analyse d'imagerie optique de la nanoparticule tumeur ciblée, Herdox. En particulier, l'utilisation de détail du dispositif d'imagerie multimode pour détecter le ciblage tumoral et l'évaluation de la pénétration tumeur est décrit ici.

Vidéo: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

Introduction à l&#39;échographie destruction des microbulles ciblées Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

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Bio-ingénierie

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Conception d&#39;un bioréacteur pour la pression cyclique Ex Vivo Étude des valves cardiaques aortiques

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

L&#39;extraction des lipides cellulaires pour ciblées dilution isotopique stable analyse de spectrométrie de masse chromatographie liquide-

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Livraison de gènes et transfection de cellules humaines de cancer du pancréas en utilisant facteur de croissance épidermique ciblant les récepteurs des base de gélatine nanovecteurs Conçu

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

Les nanoparticules virales pour<em&gt; In vivo</em&gt; Imagerie des tumeurs

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Évaluation de livraison Gene polymère nanoparticules d&#39;analyse de suivi par des nanoparticules et de cytométrie en flux à haut débit

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

Harmonique nanoparticules régénératrice recherche

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

Production de nanoparticules métalliques par laser-ablation pulsée dans les liquides: un outil pour étudier les propriétés antibactériennes des nanoparticules

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Corrélatives microscopie électronique léger (CLEM) de suivi et d’imagerie protéine virale associés Structures des cellules immobilisées Cryo

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protéine Kinase C-delta inhibiteur Peptide Formulation utilisant des nanoparticules d’or

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...